Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 207034, 10 pages doi:10.1155/2011/207034
Research Article Comparison of Adjunctive Naoxintong versus Clopidogrel in Volunteers with the CYP2C19∗2 Gene Mutation Accompanied with Qi Deficiency and Blood Stasis Constitution Hui Chen,1, 2 Guangwei Yu,1 Hong Sun,3 Xiaoying Wu,1 and Huan Wang1 1
Department of Internal Medicine, Fujian Provincial Cardiovascular Disease Institute, Provincial Clinical College of Fujian Medical University, Fuzhou, Fujian 350001, China 2 Clinical Discipline of Chinese and Western Integrative Medicine, Fujian University on Traditional Chinese Medicine, Fuzhou 350108, China 3 Department of Pharmacy, Fujian Provincial Hospital, Fuzhou 350001, China Correspondence should be addressed to Hui Chen,
[email protected] Received 18 October 2010; Accepted 6 January 2011 Copyright © 2011 Hui Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This study was to determine the impact of adjunctive Buchang Naoxintong Jiaonang (BNJ) to clopidogrel on volunteers with the CYP2C19∗ 2 gene mutation accompanied with qi deficiency and blood stasis (QDBS) constitution. Eighteen males with QDBS constitution were selected, who were 6 CYP2C19∗ 1/∗ 1, 6 CYP2C19∗ 1/∗ 2, and 6 CYP2C19∗ 2/∗ 2, and signed informed consent. Results showed that the maximal platelet aggregation (Aggmax ) and 5 min aggregation (Agglate ) with 5-µmol/L ADP in three different CYP2C19∗ 2 genotypes were significantly decreased after any drug therapy compared with corresponding baseline measurements (all values P < .05). But percent inhibitions of Aggmax and Agglate (IPAs) in CYP2C19∗ 2/∗ 2 genotype at 4 hours, 24 hours, 3 days, and 7 days after clopidogrel administration were all the lowest among three CYP2C19∗ 2 genotypes (P < .01), and IPAs in CYP2C19∗ 1/∗ 2 genotype were between CYP2C19∗ 1/∗ 1 and CYP2C19∗ 2/∗ 2. IPAs had no significant difference among three different CYP2C19∗ 2 genotypes after BNJ or adjunctive BNJ. In addition, changes of CD62P, PAC1, and sCD40L were similar to changes of ADP-induced platelet aggregation in three different CYP2C19∗ 2 genotypes. Conclusion was that adjunctive BNJ to clopidogrel can enhance the antiplatelet effect in volunteers with the CYP2C19∗ 2 gene mutation.
1. Introduction The recent US Food and Drug Administration (FDA) “boxed warning” on clopidogrel is based on the concern that the antiplatelet effect of clopidogrel depends primarily on its activation by the cytochrome P450 (CYP) system [1]. Patients with decreased CYP2C19 function because of genetic polymorphisms metabolize clopidogrel poorly and have higher rates of cardiovascular events after acute coronary syndrome (ACS) and percutaneous coronary interventions (PCIs) than patients with normal CYP2C19 function [2, 3]. CYP2C19∗ 2 is the most common genetic variant reproducibly associated with variability in clopidogrel active metabolite bioavailability, antiplatelet effects, and clinical outcomes [4, 5]. To overcome deficits in clopidogrel
responsiveness, one approach is to increase the dose of clopidogrel [6, 7]. Other strategy is to add a third drug (such as Cilostazol) to aspirin and clopidogrel to further enhance platelet inhibition [8]. The other approach is to substitute a newer, more potent platelet inhibitor drug (such as prasugrel) for clopidogrel [9]. However, these strategies that enhanced response to clopidogrel are often associated with a higher risk for bleeding that may be attributed to the overmuch inhibition of the TXA2 and ADP platelet activation pathways that are essential for normal hemostasis [10, 11]. These considerations underscore the need for agents that provide more comprehensive platelet inhibition without interfering with hemostasis, for greater protection against thrombotic events with no incremental bleeding risk. In traditional Chinese medicine (TCM), it is expressed by
2 regulating the yin and yang balance [12], by applying holistic approaches to enhance the system’s harmony, a method called dual regulation. Qi deficiency and blood stasis syndrome is the most common syndrome in patients with coronary heart disease, especially after PCI [13, 14]. Buyang Huanwu decoction (BYHWD), a TCM formula, has been recognized as a treatment for coronary heart diseases with Qi deficiency and blood stasis syndrome and cerebrovascular diseases in clinic [15]. Buchang Naoxintong Jiaonang (BNJ) consists of BYHWD plus Scorpio and Hirudo. Therefore, it was a compound preparation of BYHWD. BNJ is an approved TCM for stroke [16], which is widely used, and is well tolerated. BNJ combined with aspirin could enhance the antiplatelet effect in patients with cardio-cerebrovascular diseases [17]. The purpose of this study was to determine the impact of adjunctive BNJ in volunteers with the CYP2C19∗ 2 gene mutation accompanied with qi deficiency and blood stasis constitution during treatment of clopidogrel.
2. Methods 2.1. Subject Population. Male volunteers were eligible for enrollment if they were between 25 and 55 years of age, undergoing medical examination in Fujian Provincial Hospital, and identified as having genotype CYP2C19∗ 2. Based on the theory of constitution of TCM [18, 19], qi deficiency constitution and blood stasis (or stagnant blood) constitution were evaluated. Qi deficiency constitution means insufficiency of primordial QI; lassitude, short breath, and spontaneous perspiration are its main characteristics. Blood stasis constitution means an impeded blood flow; dark or purplish tongue and complexion are its main characteristics. Major exclusion criteria included active bleeding and bleeding diatheses, any antiplatelet or anticoagulation therapy, contraindication to antiplatelet therapy, leukocyte count 3 times upper normal, serum creatinine level >2.5 mg/dL, having no histories such as cardiovascular or cerebral vessels diseases, diabetes, phlebothrombosis, arterial thrombus, gastritis, peptic ulcer, hepatitis, cholecystitis, renal disease, neoplastic disease, external injury or operation within 6 months, inability to follow the protocol. The Institutional Review Board of Provincial Clinical College of Fujian Medical University approved the study protocol, and the patients provided written informed consent for participation. The study was performed in the Clinical Pharmacological Base of Fujian Provincial Hospital. CYP2C19∗ 2 genotyping, adenosine diphosphate- (ADP-) induced platelet aggregation, enzyme immunoassays, and flow cytometry were finished in Fujian Provincial Key Laboratory of cardiovascular disease and Fujian Provincial Clinical Laboratory, respectively. 2.2. Genotyping by TaqMan Polymerase Chain Reaction (PCR). Genomic DNA was extracted from 200 µL peripheral potassium ethylenediaminetetraacetic acid-anticoagulated blood with the TIANamp Blood DNA Kit {TIANGEN
Evidence-Based Complementary and Alternative Medicine BIOTECH (BEIJING) CO., LTD} according to the manufacturer’s instruction. CYP2C19∗ 2 (681G > A; rs4244285) was genotyped using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). MultiGene Gradient Thermal Cycler (TC9600-G-230V, Labnet International, Inc.) was selected. PCR was performed in a 25 µL reaction mixture containing 1.0 µL of DNA, 0.8 µL of each primer (forward primer 5 CAGAGCTTGGCATATTGTATC-3 and reverse primer 5 -GTAAACACACAACTAGTCAATG-3 ), 2.0 µL of each dNTPs, 2.5 µL of 10 reaction buffer, 17.6 µL of sterilizing, and 0.3 µL of Taq DNA polymerase (Xiamen Tagege Biotechnology Co., Ltd.). PCR parameters consisted of an initial denaturation for 5 min at 94◦ C, followed by 35 cycles of 30 s at 94◦ C, 20 s at 56◦ C, and 20 s at 72◦ C, and by a final extension for 5 min at 72◦ C. The PCR product was then digested with SmaI (Xiamen Tagege Biotechnology Co., Ltd.) for 5-6 hours at 30◦ C yielding 2 fragments of 212 and 109 bp in the case of the GG genotype. For the AA genotype a single band of 321 bp was observed. For the GA genotype 3 fragments of 321, 212, and 109 bp were observed. Genotypes were directly observed with an ImageMaster VDS-CL (Amersham Pharmacia Co., Sweden) and were confirmed with ABI 3700 Automated DNA Sequencer. PCR-RFLP was performed to genotype CYP2C19∗ 2 in 360 medical examination male volunteers aged 25–55. There were 204 subjects with CYP2C19∗ 1/∗ 1(681GG) genotype, 127 subjects with CYP2C19∗ 1/∗ 2(681GA) genotype and 24 subjects with CYP2C19∗ 2/∗ 2(681AA) genotype. 2.3. Study Design. The ABNJC-CYP2C19∗ 2 (Adjunctive Buchang Naoxintong Jiaonang versus Clopidogrel in Volunteers with CYP2C19∗ 2 Gene Mutation) study is a prospective, controlled platelet function study of CYP2C19∗ 2 Gene Mutation in Volunteers with Qi Deficiency and Blood Stasis Constitution. The flow diagram of the study is depicted in Figure 1. Eighteen male volunteers with qi deficiency and blood stasis constitution were selected, who were 6 CYP2C19∗ 1/∗ 1, 6 CYP2C19∗ 1/∗ 2, and 6 CYP2C19∗ 2/∗ 2, and signed informed consent. All subjects have stopped taking any medicine two weeks before the study. There were no cigarette smoking, no drinking alcohol, and no drinking coffee during the study. Each subject took clopidogrel 300 mg on the first day and then 75 mg once daily for consecutive six days. After seven washing days, BNJ (0.8 g thrice per day) was taken for five days. Then BNJ-combined clopidogrel were taken for 7 days (the dose was the same as above). BNJ (Naoxintong) 0.4 g capsules (Compilation of The National Standard of Chinese Traditional Medicine no. WS10001 (ZD-0001)-2002; Med-drug Permit no. Z20025001) were supplied by the Buchang Pharmaceutical Co., Ltd., and Plavix (clopidogrel bisulfate) 75 mg tablets (Med-drug Permit no. J20080090) were provided by Sanofi-Winthrop Industrie. The test, dispensing, and records were taken by GCP (good clinical practice) trained doctor. Venous blood samples were drawn by trained nurses. Blood samples were obtained to measure the ADP-induced platelet aggregation by turbidimetry at baseline and 4 hours, 24 hours, 3 days and 7 days after clopidogrel administration, 7 days
Evidence-Based Complementary and Alternative Medicine
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Genotype CYP2C19∗ 2 was performed in medical examination male volunteers (n = 360)
CYP2C19∗ 1/ ∗ 1 (n = 6)
CYP2C19∗ 1/ ∗ 2 (n = 6)
CYP2C19∗ 2/ ∗ 2 (n = 6)
Sampling
Baseline
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4 hours
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24 hours
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3 days
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Washing for 7 days
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BNJ 0.8 g, tid for 5 days
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Clopidogrel 300 mg on the first day and then 75 mg once daily for consecutive six days after signing informed consent
BNJ combined clopidogrel for 7 days
Time table ∗
Figure 1: Study protocol timeline. The figure shows the ABNJC-CYP2C19 2 trial protocol.
after washing, 5 days after BNJ administration, and 7 days after BNJ combined clopidogrel, and measure the platelet count, platelet functional (ADP-induced platelet aggregation, Platelet activation of the membrane marker P-selectin (CD62p), and Platelet Activator combined-l (PAC-1) with flow cytometry) and inflammatory index (Soluble CD40L (sCD40L) with enzyme-linked Immunosorbent assay) at baseline, 7 days after clopidogrel administration, 5 days after BNJ administration, and 7 days after BNJ-combined clopidogrel, respectively. Hemoglobin (Hb), Platelet count, fasting plasma glucose (FSG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), plasma total cholesterol (TC), and plasma creatinine (Cr) were remeasured at baseline and the end of the test, respectively. Creatinine clearance rate (CGCCr) was calculated by Cockcroft/Gault formula: CGCCr = (140-age)∗(Wt in kg)∗(0.85 if female)/ (72∗Cr). 2.4. Adenosine Diphosphate-Induced Platelet Aggregation. Aggregation studies were performed within 2 hours after blood collection, at 37◦ C, by using a turbidimetric method
of Born [20] on an LBY-NJ4-channel platelet aggregation analyzer (Beijing Precil Instrument Co., Ltd). The whole blood was centrifuged at 700 rpm for 4 min to prepare platelet-rich plasma (PRP). The remaining blood was further centrifuged at 3500 rpm for 10 min to prepare platelet-poor plasma (PPP). The platelet counts of PRP were adjusted to 200 × 109 /L. Results were recorded as light transmission at maximal aggregation (Aggmax ) and 5 min aggregation (Agglate ) after the addition of ADP (SIGMA-ALORICH) at final concentrations of 5 µmol/L. Aggmax is considered to reflect the activity of both P2Y1 and P2Y12 receptors, whereas Agglate is more reflective of P2Y12 receptor activity. Inhibition of platelet aggregation (IPA) was defined as the percent decrease of aggregation values (Aggmax and Agglate ) between baseline and after treatment and calculated as follows: IPA (%) = ([intensity of aggregation at baseline − intensity of aggregation after treatment]/[intensity of aggregation at baseline]) × 100 [8]. Percentage of platelet disaggregation between Aggmax and Agglate was defined as follows: disaggregation (%) = ([Aggmax − Agglate ]/[Aggmax ]) × 100 [8].
2.5. Flow Cytometry. All flow cytometric studies were conducted on Beckman Coulter Epics XL Flow Cytometer (Becton Dickinson, America) using CellQuest software (Becton Dickinson) for data acquisition and analysis. Analyses were performed on citrated whole blood diluted 1 : 9 in phosphate-buffered saline incubated with either PAC-1 fluorescein isothiocyanate-conjugated monoclonal antibody (Becton Dickinson) and anti-P-selectin (CD62p) phycoerythrin-conjugated monoclonal antibody (Becton Dickinson). Platelet activation markers (CD62p and PAC-1) were measured in unstimulated platelets and after stimulation of platelets with ADP (5 µmol/L final concentration). 2.6. Enzyme Immunoassays (EIAs). Serum levels of Soluble CD40 ligand (sCD40L, R&D Systems Inc., America) were determined by EIA (detection limit, 0.03 ng/mL; Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions (intra- and interassay coefficient of variation
